WO2017140878A1 - Arabinanase and uses thereof - Google Patents

Arabinanase and uses thereof Download PDF

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Publication number
WO2017140878A1
WO2017140878A1 PCT/EP2017/053672 EP2017053672W WO2017140878A1 WO 2017140878 A1 WO2017140878 A1 WO 2017140878A1 EP 2017053672 W EP2017053672 W EP 2017053672W WO 2017140878 A1 WO2017140878 A1 WO 2017140878A1
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WIPO (PCT)
Prior art keywords
enzyme
arabinanase
juice
preparation
activity
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PCT/EP2017/053672
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English (en)
French (fr)
Inventor
Angelika Hanreich
Joachim Pheiffer
Sebastian WORCH
Gotthard Kunze
Tobias SCHWALENBERG
Reinhard PÄTZ
Original Assignee
Biopract Gmbh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Biopract Gmbh filed Critical Biopract Gmbh
Priority to AU2017220672A priority Critical patent/AU2017220672B2/en
Priority to BR112018016297-3A priority patent/BR112018016297B1/pt
Priority to ES17706438T priority patent/ES2919825T3/es
Priority to CN201780009740.3A priority patent/CN108603184B/zh
Priority to DK17706438.3T priority patent/DK3417057T3/da
Priority to CA3010581A priority patent/CA3010581C/en
Priority to PL17706438.3T priority patent/PL3417057T3/pl
Priority to EP17706438.3A priority patent/EP3417057B1/en
Publication of WO2017140878A1 publication Critical patent/WO2017140878A1/en
Priority to US16/104,911 priority patent/US11053487B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/14—Pretreatment of feeding-stuffs with enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01099—Arabinan endo-1,5-alpha-L-arabinosidase (3.2.1.99)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
    • Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to hydrolytic enzymes with arabinanase activity and their use as feed additive in livestock breeding.
  • Arabinan is a neutral polysaccharide that mainly occurs as side chain of pectin.
  • a linear a-L- arabinan consists of a backbone of arabinose monomers linked by a-1 ,5-glycosidic (arabinofuranosidic) bonds.
  • arabinose monomers or arabinose oligomers are linked to the backbone via a-1 ,2- or a-1 ,3-glycosidic (arabinofuranosidic) bonds.
  • Arabinanases are hydrolytic enzymes capable of cleaving arabinan. Endo-arabinanases (EC 3.2.1 .99) hydrolyze a-1 ,5-glycosidic bonds within the linear backbone of a-L-arabinans. a-L- arabino-furanosidases (EC 3.2.1 .55) hydrolyze a-1 ,5- and/or a-1 ,3- and/or a-1 ,2- glycosidic bonds at the non-reducing end of a-L-arabinans or a-L-arabinose-oligomers, thereby releasing arabinose monomers.
  • Rapeseed meal (RSM) and soybean meal (SBM) are byproducts of bioethanol and food production. They are inexpensive, available on a large scale, and have a high protein content. For these reasons, they are often used as feed ingredients for poultry and pigs. They contain many non-starch polysaccharides (NSPs), mainly pectin, that can only partly be digested by the animals. For poultry, the digestability of NSPs from rapeseed meal and soybean meal is 14% and 20%, respectively. Pigs are able to digest 58% of NSPs from rapeseed meal and 84% of NSP from soybean meal. The degree of digestion of substrates also differs between animals of different age. Especially young animals cannot sufficiently use carbohydrates present in the diet, which has negative effects on growth performance (antinutritive effect).
  • NSPs non-starch polysaccharides
  • feed ingredients or compound feed can be pretreated mechanically, thermally or enzymatically.
  • enzymes in livestock feeding were established in the 1980s.
  • Prevalent enzyme preparations added to feed comprise cellulase, xylanase, glucanase and/or pectinase activity. Some preparations contain several enzyme activities (multi component preparations), others contain only one (single component preparations).
  • WO 1992017592 A1 (“Cloning and expression of DNA molecules encoding arabinan- degrading enzymes of fungal origin) describes cloning of a fungal arabinanase and its use in production of fruit and vegetable juice.
  • WO 199402061 1 A1 (An enzyme with arabinanase activity") describes an enzyme preparation enriched in an arabinanase from Aspergillus aculeatus.
  • WO 1995029598 A1 (“Enzymatic treatment of soy) describes an enzyme mixture comprising several enzyme activities (including an endo-arabinanase of fungal origin) for the treatment of an aqueous suspension of soy meal.
  • Novel arabinohydrolases describes a multi-enzyme composition comprising several enzymes with arabinanase activity for hydrolyzing arabinans present in plant biomass.
  • the problem underlying the present invention is to improve the digestability of pectin-rich feed ingredients or mixed feed.
  • the problem is solved by the subject-matter of the independent claims. Description of the invention
  • a first aspect of the invention relates to an isolated enzyme having arabinanase activity and the use of an isolated enzyme having arabinanase activity.
  • the arabinase activity is characterized in that it comprises the activities of
  • arabinose signifies a polysaccharide comprising a backbone of arabinose monomers linked by a-1 ,5-glycosidic bonds.
  • arabinose monomers or arabinose oligomers are linked to the backbone via a-1 ,2- and/or a-1 ,3-glycosidic bonds (Fig. 1 ).
  • enzyme signifies a single protein consisting of a single amino acid chain.
  • isolated enzyme signifies that said enzyme was intentionally produced/isolated and is not a random, minor or negligible component or byproduct.
  • the enzyme having arabinanase activity according to the present invention is able to degrade arabinan from the reducing end (arabinofuranosidase activity) and from within the arabinan chain (endoarabinanase activity). It is thus bifunctional.
  • the enzyme is characterized by an amino acid sequence exhibiting at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 100% identity with SEQ ID NO 02.
  • the enzyme according to the present invention was amplified from A. adeninivorans genomic DNA.
  • the enzyme additionally catalyses hydrolysis of (1 -3) and/or (1 ,2)-a- arabinofuranosidic linkages at the non-reducing end of a-L-arabinans or a-L-arabinose- oligomers.
  • the presence of both endoarabinanase activity and arabinofuranosidase activity has the advantage that branched arabinan can be degraded more efficiently.
  • the arabinofuranosidase activity degrades the arabinose oligomers linked to the arabinan backbone and removes the arabinose monomers linked to the arabinan backbone (debranching).
  • the linear backbone is degraded by the endoarabinanase activity (depolymerization).
  • Endoarabinanase activity is known to be inhibited the presence of arabinan oligomers. Due to the arabinofuranosidase activity, the oligomers produced during the depolymerization are degraded.
  • the dual activity of the enzyme according to the present invention thus results in a complete and efficient degradation of arabinan.
  • the enzyme exhibits optimum activity at a temperature between 40°C and 55°C.
  • the enzyme exhibits optimum activity at a pH between 3.5 and 5.5.
  • the enzyme according to the present invention is thermostable up to a temperature of approx. 65 °C. During incubation at 60°C for 2 hours, enzymatic activity does not decrease. This signifies an improved thermo-stability compared to the arabinanase isolated from Aspergillus aculeatus (WO 199402061 1 A1 ), which is partly inhibited at 50°C.
  • the enzyme according to the present invention is also stable between pH 3 and pH 9 and at a salt concentration from 50-100 mmol/l.
  • arabinanase activity can be further protected, e.g. by a heat protective coating that prevents loss of enzyme activity during pelletizing.
  • the enzyme according to the present invention is active under the environmental conditions within the digestive tract.
  • Another aspect of the invention relates to the use of the isolated enzyme according to any embodiment of the first aspect of the invention for the degradation of plant biomass or for the preparation of a feedstock additive.
  • the plant biomass is a pectin-rich plant biomass.
  • a feedstock additive is prepared and used in livestock feeding.
  • the plant biomass is a liquid preparation comprising pectin-containing plant material, in particular apple juice, pear juice, grape juice, orange juice, lemon juice, tomato juice, or carrot juice.
  • pectin-containing plant material in particular apple juice, pear juice, grape juice, orange juice, lemon juice, tomato juice, or carrot juice.
  • the degradation of plant biomass relates to improved degradation of substrates for biofuel production, particularly biogas and bioethanol production.
  • Another aspect of the invention relates to an isolated nucleic acid sequence.
  • This nucleic acid sequence from which the enzyme for carrying out the present invention can be expressed is also known as nucleic acid sequence.
  • a. encodes the enzyme according to the first aspect of the invention.
  • b. comprises or essentially consists of the nucleic acid sequence specified by SEQ ID NO 01 ;
  • c. comprises a sequence of at least 750, 800, 850, 900, 950, 1000, 1050, 1 100 or 1 150 basepairs able to hybridize to SEQ ID NO 01 under high stringency conditions.
  • high stringency conditions relates to hybridization conditions that require essentially all bases of a polynucleotide of 500 bp length to be paired with complementary bases of the other polynucleotide.
  • Another aspect of the invention relates to a recombinant expression vector, from which the enzyme for carrying out the present invention can be expressed.
  • This vector comprises the nucleic acid sequence as laid out above, wherein said nucleic acid sequence is operably linked to a regulatory element facilitating the expression of the nucleic acid sequence in a host cell.
  • Another aspect of the invention relates to a cell comprising the aforementioned nucleic acid sequence or the aforementioned recombinant expression vector.
  • the cell is a prokaryotic cell, particularly a cell belonging to the genus of Escherichia or Bacillus.
  • the cell is a eukaryotic cell, particularly a filamentous fungus or a yeast cell.
  • the filamentous fungus may be selected from an Aspergillus ssp. cell, particularly an Aspergillus oryzea cell or an Aspergillus niger cell, or from an Trichoderma ssp cell, particularly a Trichoderma reesei cell.
  • the yeast cell may be selected from an Arxula adeninivorans cell, a Hansenula polymorpha cell, a Pichia ssp. cell, particularly a Pichia pastoris cell or a Saccharomyces cerevisiae cell.
  • Another aspect of the invention relates to a method of synthesis of the enzyme for carrying out the invention.
  • a method of synthesis of the enzyme for carrying out the invention comprises the steps of
  • the cells are selected from Arxula adeninivorans or Hansenula polymorpha.
  • the fed-batch procedure comprises a batch phase and a feed phase, wherein the feed phase is performed at pH 6 and p0 2 40%.
  • a single component enzyme preparation comprising an enzyme having arabinanase activity is provided.
  • the arabinanase activity catalyses
  • single component enzyme preparation signifies an enzyme preparation essentially containing a single enzyme.
  • the single component enzyme preparation according to the present invention essentially contains a single bifunctional enzyme.
  • One advantage of using a "single component enzyme preparation" containing a bifunctional enzyme rather than a multicomponent enzyme preparation is that production of a single enzyme preparation is faster and more cost- efficient. The parameters for production and use can be optimized much easier. In addition, registration of multicomponent enzyme preparations for use as feed additive is much more complex and challenging than registration of a single component enzyme preparation.
  • the enzyme having an arabinanase activity is characterized by the amino acid sequence of SEQ ID NO 02 or by an amino acid sequence exhibiting at least 55%, 60%, 65%, 70%, 75% 80%, 85%, 90%, 92%, 94%, 96%, 98%, 99% or 100% identity with SEQ ID NO 02.
  • the arabinanase activity additionally catalyses hydrolysis of (1 -3)-a- and/or the (1 -2)-a-arabinofuranosidic linkages at the non- reducing end of a-L-arabinans or a-L-arabinose-oligomers.
  • the use of the isolated enzyme according to any embodiment of the first aspect of the invention is provided for the manufacture of a single component enzyme preparation for the degradation of plant biomass or for the preparation of a feedstock additive.
  • the plant biomass is a pectin-rich plant biomass.
  • a process for the degradation of plant biomass comprises the steps of
  • aqueous preparation of a plant biomass particularly selected from a liquid preparation comprising pectin-containing plant material, in particular apple juice, pear juice, grape juice, orange juice, lemon juice, tomato juice, or carrot juice, or a substrate for biofuel production, then
  • a compound feed comprising or essentially consisting of
  • compound feed relates to an animal feed comprising multiple components.
  • Non-limiting examples for such components are carbohydrate containing components, protein containing components and feed additives such as vitamins or enzyme preparations.
  • pectin-rich biomass relates to a biomass in which the cell walls contain more than 12% pectin on a dry weight basis.
  • pectin-rich biomass are soybean and soybean meal (SBM), lupins, rapeseed, rapeseed meal (RSM), other oilseeds and legumes and sugar beet
  • the compound feed comprises 5% - 50% (w/w), particularly 15% - 25% (w/w) of a pectin-rich biomass.
  • a compound feed that comprises a pectin-rich biomass and a single component enzyme preparation as specified in the respective aspect of the invention, and additionally comprises another feed component, particularly a starch-bearing feed component such as grain or maize.
  • the invention provides a compound feed having a significant percentage of pectin-rich biomass and a single enzyme preparation added to facilitate utilization of the pectin component.
  • the pectin and arabinanase components may be pelleted individually, or together with conventional compound feed components that are rich in starch (wheat, maize) and typically constitute between 50% and 95% of the compound feed fed to a farm animal.
  • the use of the enzyme according to the present invention as feed additive improves the digestability of pectin-rich foodstuff like soybean and soybean meal (SBM), lupins, rapeseed, rapeseed meal (RSM), other oilseeds and legumes and sugar beet.
  • SBM soybean and soybean meal
  • RBM rapeseed meal
  • the arabinanase acts exclusively on arabinan. It releases arabinose-polymers, -oligomers and -monomers, which can be further exploited.
  • fluidity of the feed is improved.
  • the enzyme does not act on the pectin backbone which consists mainly of polygalacturonan.
  • Pectinase preparations like Ronozyme VP (DSM) mainly act on the pectin backbone.
  • the arabinanase according to the present invention is faster and more efficient than comparable pectinase preparations in releasing arabinose-oligomers and/or -monomers from naturally occuring polymers.
  • a fast effect of feed additives is advantageous, since the retention time within the intestinal tract is only 4 to 8 hours for poultry and other monogastric animals (Enzyms in farm animal nutrition, 2 nd edition, Bedford und Partridge, 2010).
  • the concentration of said enzyme preparation is is 1 -500 mg/kg, particularly 10-400 mg/kg, more particularly 100-300 mg/kg.
  • the pectin-rich biomass is selected from sugar beet, oilseed and legume, more particularly rapeseed, rapeseed meal, lupins, soybean and soybean meal.
  • the compound feed further comprises an additional isolated enzyme, particularly phytase, protease and/or NSP-degrading enzyme at a concentration of 1-500 mg/kg, even more particularly 10-400 mg/kg, even more particularly 100-300 mg/kg.
  • an additional isolated enzyme particularly phytase, protease and/or NSP-degrading enzyme at a concentration of 1-500 mg/kg, even more particularly 10-400 mg/kg, even more particularly 100-300 mg/kg.
  • the compound feed comprises two isolated enzymes, selected from:
  • the enzyme according to the first aspect of the invention and a protease, particularly an acid stable serine protease.
  • a use of the compound feed described above in livestock feeding is provided.
  • the livestock is selected from monogastric animals, particularly poultry or pigs, more particularly poultry selected from chicken, duck and turkey.
  • the enzyme as specified according to any one of the above aspects or embodiments of the invention is used for treating pectin containing plant material, such as fruits and vegetables.
  • Certain embodiments of this aspect provide a process for treatment of a fruit mash or a liquid preparation comprising pectin containing plant material. The process comprises contacting a mash or liquid preparation of a plant material with an isolated arabinanase enzyme as specified in the first aspect of the invention, or a preparation thereof.
  • the arabinanase preparation which can also contain cellulases, glucanases, amylases and/or pectinases, is used for improvement of extractability and degradation of mash from fruit and vegetables.
  • the enzyme or the enzyme preparation is added to the fruit mash and incubated for 1-5 hours at temperatures between 10°C and 25°C in concentrations of 10-400 mg/kg, even more particularly 100-300 mg/kg mash. After enzymatic pretreatment, juice is pressed out of the mash.
  • the arabinanase preparation is used for viscosity reduction, especially in apple or pear juice. In certain embodiments, the arabinanase preparation is used for prevention of haze production.
  • Arabinanase or arabinanase preparations are added to hazy juice, which have pH values between 3 and 4.5, at temperatures between 10°C and 25°C in concentrations of 10-400 mg/kg, even more particularly 100-300 mg/kg juice and incubated for 1 to 5 hours. Juices are then cleared by the addition of diatomite, bentonite or other additives and filtered. The resulting clear juices are preserved by pasteurization.
  • the liquid preparation of plant material is fruit juice, particularly juice from apples, pears or grapes. In certain embodiments, the liquid preparation of plant material is vegetable juice, particularly juice from tomatoes or carrots.
  • Consistency and appearance of fruit juices may be adapted.
  • Products with specific properties such as clear juice, cloud stable juice or purees from fruits and vegetables, e.g. apple, berries, grape, citrus, tomatoes or carrots, may be produced using the arabinanase of the invention alone or in combination with other enzymes, such as cellulases, glucanases, pectinases and/or amylases.
  • the enzyme as specified according to any one of the above aspects or embodiments of the invention is used for treating pectin containing plant material, such as sugar beets, whole plant silages, fruit or other arabinan- containing organic substrates during biofuel production.
  • Biofuel production includes the production of bioethanol or biogas.
  • bioethanol can be enhanced by pretreating pectin-rich biomass, such as sugar beet, fruit, fruit mash and other pectin-containing substrates with the arabinanase of the invention alone or in combination with other hydrolytic enzymes.
  • the enzyme or the enzyme preparation is added to the pectin-rich biomass and incubated for 1 -5 hours at temperatures between 30°C and 60°C in concentrations of 10-400 mg/kg, even more particularly 100-300 mg/kg mash.
  • the degradation of the arabinan sidechains of pectin leads to a higher release of glucose, which results in a higher amount of ethanol after fermentation.
  • biogas production agricultural substrates such as sugar beets, whole plant silages or other arabinan-containing biomass
  • the arabinanase preparation alone or a combination of arabinanase and other NSP-degrading enzymes such as cellulases, glucanases, xylanases or pectinases, or a combination of arabinanase and a protease, such as an acid stable serine protease, are added to the process regularly, e.g. daily together with the substrate or via a separate supply system.
  • Biogas fermentation takes place at temperatures between 30°C and 60°C. Hydraulic retention times lie between 30 and 150 days.
  • the amount of added enzyme preparation depends on the amount of substrate fed to the fermenter. Dosages of 1 -500 mg/kg substrate, more particularly 10-400 mg/kg substrate, even more particularly 100-300 mg/kg substrate are required.
  • arabinanase of the invention alone or in combination with other enzymes, such as cellulases, glucanases, pectinases, xylanases or proteases, leads to a decrease of viscosity and/or to a higher biogas production from pectin-rich substrates under anaerobic conditions.
  • Figure 1 shows a schematic drawing of arabinan.
  • FIG. 1 shows the fed-batch procedure.
  • FIG. 3 shows the degradation of soybean meal by the arabinanase according to the present invention and by established feed additives.
  • Figure 4 shows the degradation of rapeseed meal by the arabinanase according to the present invention and by established feed additives.
  • Figure 5 shows the degradation of soybean meal by the arabinanase according to the present invention and by the arabinananse of A. niger.
  • Figure 6 shows the degradation of rapeseed meal by the arabinanase according to the present invention and by the arabinananse of A. niger.
  • arabinanase gene (AABN) was amplified from A. adeninivorans genomic DNA with the oligos
  • coli XI1 -Blue cells (recA 1 endA 1 gyrA96 thi-1 hsdRU supE44 relA 1 lac [F ' proAB lacl q ZAM15 Tn 70(Tet r )]) were used for standard heat transformation (Green, Molecular Cloning: A Laboratory Manual (Fourth Edition). 2012: Cold Spring Harbor Laboratory Press) and grown in LB medium, supplemented with ampicillin (50 pg mL ⁇ 1 ; AppliChem, Germany) when required for selection.
  • This plasmid served as template to amplify the AABN gene equipped with the constitutive TEF1 promoter with the oligos 5'- CTGCCGCGGC TCGACTTCAA TCT-3' (SEQ ID NO 05) and 5'- CCGCCGCGGC CCCAGCTTGC ATG-3' (SEQ ID NO 06), both containing Sacll restriction sites for integration of the construct into the Xplor® 2 expression/transformation vector (Boer, Appl Microbiol Biotechnol, 2009. 84(3): p. 583-594) applying the same PCR conditions as above.
  • the AABN expression cassette consisting only of yeast sequences including the ATRP1 selection marker was transformed into the auxotrophic mutant A.
  • adeninivorans G1212 [aleu2 atrpl :ALEU2] (Steinborn, J Biotechnol, 2007. 127(3): p. 392-401 ) according to the Hollenberg transformation procedure (Dohmen, Yeast, 1991 . 7(7): p. 691 -692).
  • the estimated molecular weight of the monomer is 42326.16 Daltons. After removal of the putative 23 amino acid secretion signal the mature monomer has a molecular weight of 40094.45 Daltons.
  • the enzyme exhibits endo-arabinanase activity and arabinanofuranosidase activity (EC 3.2.1 .99, EC 3.2.1.55).
  • Endo-arabinanase activity is determined in a photometric assay.
  • Linear arabinan e.g. arabinan debranched Megazyme P-DBAR
  • the enzyme is incubated with the substrate in a buffer having a certain pH (pH 4, 5, 6, 7 or 8) for a certain time (10, 20 or 30 min) at a certain temperature (40, 45, 50, 55 or 60°C).
  • a buffer having a certain pH (pH 4, 5, 6, 7 or 8) for a certain time (10, 20 or 30 min) at a certain temperature (40, 45, 50, 55 or 60°C).
  • the reducing ends of the monosaccharides and oligosaccharides released from the substrate by the enzyme are detected in a redox reaction.
  • the results are compared to a calibration curve, which represents a linear relation between several known arabinose concentrations and the absorption wavelength.
  • the endo-arabinanase activity within the enzyme preparation was determined using the substrate arabinan debranched (Megazyme) in actetate buffer having a pH of 5.0 at 55°C. The activity was 3000-5000 U g "1 preparation depending on the batch. Dry products and enzyme solutions in glycerol (10%) are stable for several months.
  • Arabinofuranosidase activity is determined using the synthetic substrate p-nitrophenyl-a-L- arabinofuranoside.
  • the enzyme is incubated with the substrate in a buffer having a certain pH (pH 4, 5, 6, 7 or 8) for a certain time (10, 20, 30 or 40 min) at a certain temperature (40°C).
  • the enzyme releases p-nitrophenol from arabinose, resulting in a photometrically detectable change in absorption at 415nm. Evaluation occurs using a p-nitrophenol calibration curve.
  • Arabinofuranosidase activity within the enzyme preparation was determined using the substrate p-nitrophenyl-a-L-arabinofuranoside (1 mM) in citrate buffer having a pH of 5.0 at 40°C. The activity measured was 200-400 U g "1 preparation depending on the batch.
  • Enzymatic activity is determined according to the following formula:
  • the arabinanase is thermostable up to a temperature of approx. 65°C. Enzymatic activity does not decrease during incubation at 60°C for 2 hours or during incubation at a pH between 3 and 9 for 48 hours.
  • the enzyme exhibits optimum activity at a temperature between 40°C and 55°C and at a pH between 3.5 and 5.5. Temperature optimum was determined in citrate buffer having a pH of 5.5. The pH optimum was determined using acetate buffer (pH 3-6.5), citrate buffer (pH 3.5-7), phosphate buffer (pH 5.5-7.5) and Tris HCI (pH 7-9). The presence of manganese-salts (MnS0 4 , MnCI 2 ) had positive effects on enzyme activity.
  • A. adeninivorans comprising the arabinanase (AABN) expression cassette is fermented in a fed-batch procedure.
  • Feed 1 Feed 1
  • a feeding loop starts (Fig. 2). Fermentation occurs at 40% p0 2 and pH 6 for 5 days; pH is adjusted using NaOH (25%) and H 3 PO4 (10%). At the end of the fermentation process, enzyme activity within the supernatant is 80 - 90 U g "1 . Yeast cells are removed in a centrifugation step and the enzyme is purified from the supernatant.
  • the degree of plant biomass degradation is determined in relation to time, temperature, pH and enzyme concentration.
  • the buffer contains sodium azide (0,02%) and antibiotics (penicillin: 100 I.U. ml "1 , streptomycin: 0, 1 mg ml "1 ), thus preventing growth of microorganisms and ingestion of the sugar moieties released by the test enzymes.
  • 140 ⁇ water is added to substrate controls, while 140 ⁇ enzyme preparation is added to the samples. Incubation occurs at 40 °C with shaking. Analyzed timepoints are 2 hours (TP 1 ), 4 hours (TP 2) and 6 hours (TP 3. At each timepoint, the enzymartic reaction is stopped by adding 140 ⁇ NaOH (2M) and 140 ⁇ glucose (30mM). Samples are then centrifuged for 5 min at 10.000 g.
  • the amount of glucose equivalents (in mg) released by the enzymatic reaction is determined by comparison of the samples with the glucose standard curve.
  • the degradation activity of the enzyme is determined by the ratio of the released reducing sugars and the deployed substrate (50 mg plant biomass, VS).
  • the inventors show that by combined addition of the arabinanase according to the present invention and a pectinase product, a synergistic effect can be achieved.
  • Ronozyme Multigrain containing mainly xylanase and glucanase
  • Ronozyme VP containing hemicellulases and pectinases
  • the degradation of RSM by the arabinanase according to the present invention was approx. 10% after 6 hours (Fig. 4).
  • the inventors show that by combined addition of the arabinanase according to the present invention and a pectinase product, a synergistic effect can be achieved.
  • broilers were fed with a compound feed diet supplemented with the arabinanase of the invention. At 10 days of age, 96 broilers were divided into 4 groups. All broilers received a basal diet comprising rapeseed meal (35%) and corn (60%). Group A received the basal unsupplemented diet, group B received the basal diet supplemented with an established feed enzyme product (Ronozyme VP) and groups C and D received the basal diet supplemented with the enzyme according to the present invention (Tab. 2). Duration of the feeding experiment was 15 days. During the adaption phase (day 1 1-14) all groups received the basal diet without any enzyme supplementation.
  • the endo-activity of the arabinanase of the invention (Arxula adeninivorans) and of a commercial arabinanase from Aspergillus niger (Megazyme E-EARAB) were determined in a photometric assay.
  • Debranched arabinan (Megazyme P-DBAR) was used as substrate.
  • the enzymes were incubated with the substrate in a buffer having a pH of 5 for 20 min at 55 °C. At the end of the incubation time, the reducing ends of the monosaccharides and oligosaccharides released from the substrate by the enzyme were detected in a redox reaction.
  • the activity of the arabinanase of the invention was 5500 U/g and the activity of the commercially available Aspergillus niger arabinanase was 645 U/g.
  • Plant biomass degradation induced by the two enzymes was determined in an abiotic test as explained in detail above. Rapeseed meal and soybean meal were used as insoluble substrates, because these substrates contain high amounts of pectin, which is rich in arabinan side chains.

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ES17706438T ES2919825T3 (es) 2016-02-18 2017-02-17 Arabinanasa y usos de la misma
CN201780009740.3A CN108603184B (zh) 2016-02-18 2017-02-17 阿拉伯聚糖酶及其用途
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PL17706438.3T PL3417057T3 (pl) 2016-02-18 2017-02-17 Arabinanaza i jej zastosowania
EP17706438.3A EP3417057B1 (en) 2016-02-18 2017-02-17 Arabinanase and uses thereof
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